Method and apparatus for photoanalysis

ABSTRACT

A narrow beam of monochromatic light is directed through an optical chamber to intersect a thin stream of small particles to be optically analyzed, causing the particles to emit fluorescent radiation in the presence of a significant particle characteristic to be detected. A spectral filtering element is positioned to intercept the fluorescent particle radiation and to pass only a selected wave length to a photoresponsive pick-up element for detection thereby.

United States Patent Friedman'et al.

METHOD AND APPARATUS FOR PHOTOANALYSIS Inventors: Mitchell Friedman,Yorktown Heights; Louis A. Kamentsky,

Briarcliff Manor; Isaac Klinger, Yorktown Heights, all of N.Y.

Assignee: Bio/Physics Systems, Inc., Katonah,

Filed: Dec. 6, 1971 Appl. No.: 205,434

Related US. Application Data Division of Ser. No. 2,750, Jan. 14, 1970,Pat. No, 3,687,553.

US. Cl 356/39, 356/102, 356/104, 250/458 Int. Cl. G0ln 33/16, GOln 21/00Field of Search..... 356/39, 102, 103, 173, 186, 356/201, 104; 250/71 RReferences Cited UNITED STATES PATENTS 11/1968 Kamentsky 356/39 Jan. 29,1974 3,497,690 2/1970 Wheeless, 11. etal 250/71 2,816,479 12/1957 s16an..356/102 3,361,030 1/1968 Goldberg ..356/103 3,566,114 2/1971 Brewer250/71 Primary Examiner-David Schonberg Assistant Examiner-Conrad ClarkAttorney, Agent, or Firm-Curtis Ailes ABSTRACT A narrow beam ofmonochromatic light is directed 7 through an optical chamber tointersect a thin stream of small particles to be optically analyzed,causing the particles to emit fluorescent radiation in the presence of asignificant particle characteristic to be detected. A spectral filteringelement is positioned to intercept the fluorescent particle radiationand to pass only a selected wave length to a photoresponsive pick-upelement for detection thereby.

13 Claims, 7 Drawing Figures 7 PATENTEDJANZQ mm 3788' 744 I SHEET 1 0F 4PATENTEUJANZQ 1914 3. 788,744

SHEEI 3 0F 4 FIG. 3 42 6 Mil.

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PATENTEDmzs mm 3,788; 744

saw u or 4 FIG. 5 90% METHOD AND APPARATUS FOR PHOTOANALYSIS This is adivision, of application Ser. No. 2,750 filed Jan. 14, 1970 and now US.Pat. No. 3,687,553 issued 8/29/72.

The present invention relates to photoanalysis apparatus, and moreparticularly to photoresponsive apparatus for detecting variouscharacteristics of small particles such as blood cells.

There is a great need for accurate analysis of the characteristics ofgroups of small particles such as in the analysis of air pollution andwater pollution conditions. A particularly important field for suchanalysis is in medical research and diagnosis. For this purpose, bloodcells and other biological cells must be analyzed.

In the present invention the analysis of small particles is accomplishedoptically by entraining the particles in a very thin stream of liquid sothat the particles pass one by one in the stream through an opticalscanning station. A photo-optical detecting device is arranged to detectthe optical reaction of each particle to illumination from a beam oflight. This method has been shown to impart very important informationabout the particles which have been scanned. The information derived isparticularly valuable when special procedures are followed forpreliminary preparation of the particles, such as the prior applicationof dyes which are taken up by the particles in different ways related tothe differences in particles which are to be detected. The particleshaving these differences may be differentially counted.

In accordance with one aspect of the present invention, it has beendetermined that in an apparatus of the above description, the value ofthe information which can be derived photo-optically is tremendouslyincreased if at least two different optical reactions of the particlescan be detected simultaneously by means of optical pick-up elementsarranged at different angular positions with respect to the direction ofthe beam of light directed at the particle.

One of the most useful optical reactions to the illumination is ameasurement of the optical absorption of the particles. The amount ofoptical absorption of the particles may be directly related to theamount of dye which has been taken into each particle in a preliminaryconditioning step. Since the dye uptake by the particles may bedifferent based upon different characteristics, the optical absorptionmay thus be used to distinguish these characteristics. The combineddetection of absorption and another optical reaction, such as scatter,therefore provides a very useful combination of information about theparticles under observation. For instance, in the analysis of biologicalcell samples, such as blood cells, it is recognized that dead cellsabsorb the dye Trypan blue while live cells do not. Accordingly, it ispossible to analyze a sample of such cells by first exposing the sampleto such a dye and then optically measuring both absorption and scatter.The dead cells which take up the stain are then optically detected bythe absorption signal, and the live cells are detected by the opticalscatter signal, and the apparatus may be used to count and record thenumbers of dead and live cells in the sample.

Accordingly, it is an object of the present invention to provide amethod and an apparatus in which at least two different opticalreactions of particles can be detected, and more specifically in whichsidderent conditions of individual cells, such as the differentiation oflive and dead cells may be accomplished reliably and quickly.

In photoanalysis apparatus of the above description, it has beenrecognized that the light scattering effect produced by particles underobservation varies according to different characteristics of theparticles, including such factors as particle size, refractive index,and the presence of refractive and absorbent substances in theparticles. Accordingly, the detection of scattered light from theparticles provides an extremely valuable method for determining thecharacteristics of the particles. Furthermore, the magnitude of thescatter radiation as a function of the angles or the ranges of anglesover which the scattering of light occurs provide distinctiveinformation. In apparatus heretofore available, it is generally beenpossible to detect light scattered by particles under observation onlyfor a very small fixed range of scatter angles. This has beenaccomplished by projecting illumination to the particle by means of alens having a mask over the central portion thereof to create a cone ofdarkness" beyond the particle positioned at the focal point of the lens.The scatter illumination is detected within this cone of darkness.

In accordance with another aspect of the present invention, it isanother object of the present invention to provide a photoanalysisapparatus for optical analysis of particles in which very accuratemeasurements of scatter illumination can be made at any desired angle,or over any desired range of angles, without specific limitation to aparticular cone of darkness.

In carrying out the above object, a structure is employed in which thereis produced an extremely narrow beam of illumination directed at theparticles, and photosensing devices are arranged at positions displacedfrom the beam to receive illumination scattered at predetermined anglesfrom the beam by the particles.

Another problem in photoanalysis apparatus of the above description hasalways been the economical production of a suitable optical chamberthrough which the particles to be examined are passed and subjected toillumination. It has been previously thought to be absolutely essentialto provide flat sides of this optical chamber in order to avoiddistortion of the light beam directed through the chamber by thematerial of the side walls of the chamber.

Accordingly, it is another object of the present invention to provide avery satisfactory and easily produced optical chamber in a photoanalysisapparatus which is extremely economical to produce because of the mannerin which provision is made for satisfactory optical properties of theside walls.

In carrying out the above object of the invention, photoanalysisapparatus is provided having an optical chamber in the form of acylindrical tube.

Further objects and advantages of the invention will be apparent fromthe following description and the accompanying drawings.

In carrying out the invention in one preferred form thereof, there isprovided an apparatus for simultaneous optical measurement of severalcharacteristics of each particle of a group of small particles such asblood cells while the particles are suspended in a liquid, including asource of light, a housing comprised of a material which transmits lightfrom the source and defining an optical chamber. A means is provided formoving the particle suspending liquid through the housing in a thinstream to convey the particles in sequence through the stream one byone. Another means is provided for directing light from the light sourceinto one side of the housing to intersect with the thin stream ofparticles in a narrow beam, and at least two photoresponsive pick-upelements are positioned outside of said housing at different angularpositions with respect to the direction of the beam when measured fromthe intersection of the beam with the stream of particles, thephotoresponsive pick-up elements being effective to simultaneouslydetect different optical reactions of each particle to illumination fromthe beam.

In the accompanying drawings:

FIG. 1 is a schematic top view, partly in section, of the most essentialelements of a photoanalysis apparatus in accordance with the presentinvention.

FIG. 2 is a sectional side view of a portion of the apparatus shown inFIG. 1 and illustrating additional photooptical pick-up elements fordetecting scatter at additional angles.

FIG. 2A is a sectional side view corresponding to FIG. 2 andillustrating, on a reduced scale, an alternative arrangement of theapparatus including means for picking up and detecting fluorescence ofthe particles.

FIG. 3 is an enlarged sectional front view taken through a section atthe center of the optical chamber of the apparatus of FIG. 1, andillustrating the intersection of the beam of light with the particlesbeing measured.

FIG. 4 is a partial front view of a photoanalysis apparatus inaccordance with the invention and having modified photoelectric pick-upelements for the detection of scattered illumination.

FIG. 5 is a top view, corresponding to FIG. I, but illustrating amodification of the photoresponsive pickup elements in which separatesignals are derived to indicate the intensity of scattered radiation anthe angle of the scattered radiation.

And FIG. 6 is a top view of two optical chamber tube members for aphotoanalysis apparatus in the course of production, in accordance witha preferred method of production.

Referring more particularly to FIG. 1, there is shown an optical chamberformed by a glass tube member 10 clamped between metal members l2, 14,which respectively include liquid tight annular seals 16 and 18. The

liquid 19 containing the particles to be observed enters the apparatusthrough a tube 20 centrally disposed within member 12. Another liquid23, which forms a sheath for the liquid l9conta'ining the particlesenters the member 12 through an entrance opening 22. The liquids cometogether in the cone or funnel-shaped entrance portion 24 of the centralbore 26 of the cylindrical member 10.

The velocity and volume of flow of the particlebearing liquid 19entering through tube 20 and the other liquid 23 entering throughentrance 22 are such as to cause the stream of particle-bearing liquidto be narrowed down at the end of the tube 20, as shown at 28, into avery narrow stream 29 having a maximum dimension of the same order ofmagnitude as the maximum dimension of the particles being carried by thestream. For instance, this dimension may be in the order of a micronstream diameter. The particle of greatest interest are often somewhatsmaller than this, being in the range from 1 to 10 microns in diameter.The liquid 23 may be referred to hereinafter as the sheath flow liquidsince it forms a liquid sheath about the narrowed stream 29. In order toprovide a smooth and non-turbulent flow of the sheath liquid 23, two ormore radial inlet openings 22 may be provided to the central bore of themember 12. The funnel-shaped entrance portion 24 of the cylindricalmember 10 is preferably provided with a special exponential functionshape, as described more fully below, particularly in connection withFIG. 6, in order to provide for smooth non-turbulent flow of the liquidsat the critical position 28 where the particle-carrying liquid isnarrowed down. Typically, the particle-carrying liquid may be an aqueoussolution and the sheath liquid 23 may be water.

The stream 29 of particles is illuminated by a beam of light emitted bya light source 30 which preferably consists of a laser. One satisfactorylaser, for instance, is a helium-neon laser. The beam oflight from thelaser is reduced in diameter by a combination of spherical lenses 32 and34. The resultant reduced diameter beam is collimated. This concentratedbeam is narrowed by a lens 36 to provide a very narrow beam at the point38- where the beam intersects with the stream 29 of particles underobservation. For this purpose, the lens 36 is preferably a cylindricallens having its cylinder axis arranged in a plane perpendicular to theaxis of the chamber cylinder 10. Thus, the pattern of the illuminationof the beam at the point 38 where it strikes the'stream of particles isa very narrowellipse which appears to be a thin line of light transverseto the stream of particles. This will be described more fully below inconnection with FIG. 3.

Electrical photoresponsive pick-up elements are arranged around theoutside of cylindrical chamber member 10 to detect different opticalreactions of each particle to illumination from the beam through lens36. For instance, an electrical photo-responsive pick-up element 40 isarranged in direct line with the beam to measure the absorption ofillumination by each particle. The resultant electrical signlas areconnected to apparatus schematically shown at 46 for amplification andrecording or display; In the absence of a particle at the intersectionof the beam, or in the absence of any substantial absorption, the beamstrikes the element 40 without any substantial diminution.

As illustrated in the drawing, the beam iverges to a certain extentafter having been converged at the center of the chamber at 38. Theeffective divergence in a practical embodiment has been limited toapproximately one degree on each side of the center line of the beam asmeasured from the particle scanning point 38 at the center of thechamber. Thus, photoresponsive pick-up elements 42 and 44 are arrangedon opposite sides of the direct beam and can be used to measureillumination scattered out of the direct beam by the particles over aselected range of angles from one degree up to a predetermined angularlimit. For instance, this range of angles may be from one to ninedegrees. As shown in the drawing, the photoresponsive pick-up elements42 and 44 may be electrically connected in parallel so that electricalsignals resulting from illumination scattered on either side of the beamwill be detected and may be recorded by the electrical apparatusschematically illustrated at 46. Additional pairs of photoresponsivepick-up elements for detecting scattered light at other ranges of anglesmay be provided as shown at 47 and 48. For instance, this additionalpair of pick-up elements may detect scatter over the scatter angle rangefrom 9 to 22. The wider angle scatter sensors 47 and 48 may be employedfor the purpose of providing a measurement of optical absorption of theparticles as an alternative to the absorption measurement by pick-upelement 40. It is known that abosrption of the incident beam willdecrease the amount of light scattered by the particles. This decreasein scattered illumination is more pronounced for the wider anglescatter, than for the near forward scatter de' tected by sensors 42 and44. Accordingly, it has been found to be advantageous to use a widerangle scatter measurements at 47 and 48 to detect absorption becausenoise signals due to light source intensity fluctuations and flow streamvibrations are much less for the scatter sensors than for the directmeasurement absorption sensor 40 which is in the direct path of thebeam.

Scattering of illumination from the particles in the reverse direction,called back-scattering, can also be detected by photoresponsive elements50 and 52 arranged on the same side of the chamber as the light source30 and connected in parallel to an electrical pick-up and recordingapparatus 54. It will be understood that the electrical apparatus 54 maybe combined with the apparatus 46, but it is shown separately here tosimplify the drawing.

The apparatus 46 and Y54 may include amplifiers, logic circuitry,digital counters, and electronic display devices. It is one of theimportant features of the invention that different optical reactions ofeach particle to illumination may be detected, processed, and recordedsubstantially simultaneously. The relationships between these differentoptical reactions may be processed by analog and digital circuitry,displayed, recorded, and plotted as a basis for making detaileddeterminations about the particles, differentially classifying theparticles, or determining the frequency with which the particularcharacteristics appear in successive particles. Because of the uniquefeatures of this invention, particle analysis and counting rates in theorder of ten thousand particles per second may be achieved. It will beunderstood that this speed is well within the capacity of the electronicand digital portions of the system.

The photoresponsive pick-up elements, such as elements 42 and 44 for thedetection of scatter, are illustrated in FIG. 1 as though they werefixed with relation to the cylindrical chamber member 10. However, suitable means is provided for precisely changing the position of thosepick-up elements in relation to the beam from source 30. This adjustmentmay be an adjustment from side to side and it may also be an adjustmentto place the elements in greater or lesser proximity to the scanningposition 38. By moving the elements away from the scanning position, theinner margins of the elements may be precisely positioned with respectto the outer margins of the direct radiation beam directed to theabsorption pick-up element 40. Thus, the elements 42 and 44 are capable,when so adjusted, of picking up scatter radiation over the narrowestpossible angle outside of the direct radiation beam path.

FIG. 2 is a partial sectional side detail view of the apparatus of FIG.1 including the cylindrical chamber member 10, the cylindrical lens 36,the absorption and scattering pick-up elements 40. 44. and 48. and thehack-scattering pick-up element 52. As illustrated in FIG. 2, thecylindrical shape of the chamber member 10 causes a refractive effectupon the light beam supplied through the cylindrical lens 36 whichcauses the light beam to converge towards the center bore 26 of thechamber member 10. This effect is shown in an exaggerated form in FIG.2. The diamter of the beam as it enters the cylindrical lens 36 isactually selected so as to be approximately equal to the diameter of thecenter bore 26. This diameter is ofthe order of 250 microns. However,the convergence of the beam in the plane of FIG. 2 (perpendicular to theaxis of the chamber member 10) is not a disadvantage since it serves toconcentrate the beam in the central portion of the center bore 26 wherethe particle carrying strea is located. Since the particle carryingstream has a diameter of only about 25 microns, a considerableconvergence of the beam is desirable. This provides relatively uniformillumination over the diameter of the particle-carrying stream, eventhough the original energy distribution from the laser beam isnon-uniform. Furthermore, if the beam were not caused to converge uponthe center bore 26, the outer portions of the beam would strike theinterface between glass and liquid at the center bore 26 at an anglegreater than the critical angle of refraction so that those outerportions of the beam would be reflected away from the center bore, andlost, without passing through the liquid,

FIG. 1 illustrated how pairs of photoresponsive pickup elements such as42 and 44, and 46 and 48, can be arranged in positions spaced away fromthe primary beam in directions parallel to the axis of the cylindricalchamber member 10 for detecting small angles of scattered illumination.However, as shown in FIG. 2, when larger angles of scatter are to bedetected, the angular displacement of pick-up elements can be around thecircumference of the cylindrical chamber member. Thus, as shown in FIG.2, a pair of pick-up elements 56 and 58 may be circumferentiallyarranged to detect light scattered in a range at about 45 degrees fromthe particle scanning point 38. Similarly, pick-up elements 60 and 62may be provided to detect scatter in a range near QQ degrees. It will beunderstood that these arrangements of pick-ups are by way ofillustration only. Particular analyses will require the detection ofscatter for particular ranges of angles. The important principleillustrated by FIGS. 1 and 2 is that light scattered by particles underanalysis can be detected for any selected ranges of scatter angles fromessentially 1 up to 179 with the analyzer configuration as illustrated.

All of the components illustrated in FIG. 2, with the exception of thecylindrical lens 36, are preferably mounted upon and movable with asupport block schematically shown as a box 55 pivotally supported on afixed mounting at 57. The support block 55 may be vertically adjusted byrotation about the pivot 57 by means of a thumb screw 59 engaging thelower edge of the block 55. Thumb screw 59 is threadedly engaged withina fixed support 61. The purpose for this vertical adjustment is toprecisely position the chamber 10 with respect to the light suppliedfrom the light source through the cylindrical lens 36. If the light beamis not vertically centered upon the center bore 26 of the chamber 10, sothat the beam accurately intersects with the stream 29 ofparticle-carrying liquid, then the device may be inoperative. Theaccurate positioning of the chamber with respect to the beam is veryimportant because an offset in the positioning oithe chamber withrespect to the beam causes undue loss of beam cuergy through excessiverefraction of beam energy at the bore 26.

In the embodiment of the invention illustrated in FIGS. 1 and 2, thereis unavoidably a certain amount of radiation which is refelcted radiallyoutwardly from the scanning point 38 in a narrow ring which is confinedto a longitudinal dimension along the cylinder of the cylindricalchamber member 10 generally corresponding to the width of the enteringbeam of illumination from source 30. Thus, the scatter detectors arealways longitudinally displaced out of this ring of radiation andpreferably arranged in pairs on opposite sides of the direct radiationposition as shown by detectors 42 and 44 in FIG. 1. Similarly, in FIG.2, the scatter detectors 56, 58, 60, and 62 each represent a pair ofdetectors preferably arranged on opposite sides of the ring ofradiation.

The preferred photoresponsive pick-up elements to be employed in thepresent invention as thus far described may consist of silicon barrierlayer photo diodes. These devices are photo-voltaic devices commonlyreferred to as silicon solar cells. Suitable devices of this kind areavailable from many commercial sources.

FIG. 2A corresponds generally to FIG. 2, but illustrates alternativearrangements of the compounds, and also additionally illustrates anarrangement for picking up and detecting fluorescence of the particles.The light from the light source is again supplied through thecylindrical lens 36 to the optical chamber 10, and absorption isdetected by photo detector 40. In this embodiment of the invention, thescatter detector 44 includes an optical reflector which may consist of amirror 44B, and a photo electric device 44C arranged to receive thescatter radiation reflected by the optical reflector 448. Since thesetwo elements accomplish the same combined function as the scatterpick-up element 44 of FIG. 2, the combination of the two elements 448and 44C may bereferred to collectively as a photoresponsive pick-upelement. As explained above in connection with FIG. 1, the scatterpick-up element 44 is preferably paired with another scatter pick-upelement 42, the combination of the two pick-up elements being effectiveto detect scatter on the two sides of the light beam passing through thescanning point. By means of the reflective arrangement shown anddescribed in connection with FIG. 2A, it is possible to employ twoseparate reflectors on opposite sides of the beam, both reflectorsdirecting scatter radiation to a single photoelectric device such as44C. By-this means, the scatter radiation signals from the two sides ofthe beam need not be electrically combined. They are, instead, opticallycombined by being directed by separate reflectorsto the singlephotoelectric device.

The particular arrangement thus far described, with the reflector 44B,and a possible additional reflector, reflecting scatter signals to asingle photoelectric device 44C may be employed in the embodimentpreviously shown and described in FIG. 2, and is not necessarily limitedto combination with other features of FIG. 2A described immediatelybelow.

One of the most useful categories of optical measurement available inthe photoanalysis of particles is the measurement of fluorescence of theparticles in response to the primary radiation by the light beamdirected through the cylindrical lens 36. The particles may be stainedwith dyes so that when excited with light they are caused to emitfluorescent radiation at one or more wave lengths different from thewave length of the primary light beam. The intensity of fluorescentradiation at various wave lengths is an extremely useful indication ofthe properties and characteristics of the particles. Since thefluorescent radiation is emitted in virtually all directions from theparticle, the fluorescent radiation is gathered by the reflectors 63 and65 and generally directed, through a cylindrical lens 67, to a dichroicmirror 69. As is well known, the dichroic mirror 69 is designed toreflect radiation having a wave length shorter than a predeterminedlimit such as 5,500 angstroms and to transmit radiation having a longerwave length. The reflected radiation is directed through a filterelement 71 to a first photo multiplier tube 73. The transmittedradiation is directed through a second optical filter 75 to a secondphotomultiplier tube 77. By means of the combination of the dichroicmirror 69 and the optical filters 71 and 75, each of the photomultiplier tubes 73 and 77 receives only that light at the wave lengthpredetermined by the respective filters. If desired, additional dichroicmirrors may be provided to split up the fluorescent radiation intoadditional spectral components in order to obtain additional opticalanalysis information.

The reflective elements 63 and 65 generally extend axially along thetubular chamber member 10. However, these reflective elements arepreferably split similarly to the scatter-sensing devices as pictured inFIG. 1, so as to avoid reflecting and transmitting the ring of radiationwhich is discussed immediately above. Avoiding the reflection andtransmission of the ring of radiation is particularly important whendealing with radiation at the green end of the visible spectrum becauseit is difficult to provide efficient optical filters 71 and 75 at thatend of the spectrum. However, when dealing with fluorescence at the redend of the visible spectrum, efficient filtering is available and it isthen preferably not to provide split reflective elements 63 and 65 andto gather the ring of radiation, which includes fluorescence signals atthe desired wave length.

The reflector 63 is preferably arranged so that fluorescent radiationemitted through the lower surface of the chamber member 10 is reflectedup to the upper reflector 65. The upper reflector 65 is preferablyarranged so that all of the radiation directed to that reflector isultimately reflected to and gathered by the left portion of thatreflector and directed through lens 67, down to the dichroic mirror 69.

When making fluorescence measurements, it has been found quite effectiveto employ for the light surface 30 an argon laser which emits light inthe blue part of the spectrum. Light of this wave length has been foundto provide a high degeee of fluorescence emission by biologicalparticles which are often under oabservation. Since the particlesfluoresce in the forward" direction, it has been found to be quitedesirable to provide a filter 79 which permits the passage only of theargon blue direct radition to the scatter sensor 44B-44C, and to theabsorption sensor 40.

It will be appreciated that the arrangement shown in FIG. 2A isparticularly useful because it permits simultaneous detection of atleast four different optical reactions of a particular particlesimultaneously. Thus, absorption is detected at 40, scatter is .detectedat 448-44C, and two different wave lengths of fluorescent radiation aredetected at photo multipliers 73 and 77. Metachromatic, or combinationsof fluorescent dyes are available which, when used to stain cells, willfluoresce at two wave lengths with each fluorescent intensityproportional to a given cellular constituent.

FIG. 3 is an enlarged detail sectional front view of that portion of theapparatus of FIG. 1 where the light beam actually strikes the stream ofparticles at point 38 in the chamber 10. The stream of particles 29 isshown to contain actual entrained particles 64 which are transported oneat a time through the elliptically shaped beam of light at 66. Becauseof the elliptical shape of the beam of light, the stream 29 carrying theparticles 64 may vary in its position within the chamber without havingthe stream actually move out of the path of the beam. Thus, the beamalways intercepts the entire stream of particles. On the other hand, thenarrow elliptical shape of the beam, in which the width of the beam isof substantially the same order of magnitude as the diameter of theparticles, means that each particle causes an optimum optical reactionwith the beam. Furthermore, even if the particles are closely spacedtogether, it is virtually impossible for more than one particle to beacted upon by the beam at any par ticular instant.

FIG. 4 is a sectional front view illustrating a modification of theapparatus of FIG. 1 in which arcuate shaped pick-up elements 42A, 44A,and 46A, 48A are employed. This view is taken at a section correspondingto the section line indicated at 44" in FIG. 2, although it will beunderstood that the actual modified structure of FIG. 4 is notillustrated in FIG. 2. The scatter pickup elements 42 and 44 and 46 and48 of FIG/1 are arranged to intercept scatter over selected ranges ofangles simply by virtue ofa displacement in position along the axis ofthe cylindrical chamber member 10. As illustrated in FIG. 2, for largerscatter angles, the measurements can be made by circumferentialdisplacement of the pick-up elements about the cylindrical chamber.However, a combination of these techniques is possible in which a highefficiency is achieved by means of the semicircular pick-ups illustratedat 42A, 44A, and 46A, 48A in FIG. 4. Thus, the illumination scatteredover a particular range of angles may be scattered anywhere within apossible cone of scatter radiation terminating in a substantiallycircular radiation area such as the area defined by the pick-up elements46A and 48A. Thus, the semicircular pick'up elements are particularlyefficient in intercepting substantially all of the scatter radiationover the range of scatter angles for which they are designed.

FIG. is a top view corresponding to FIG. 1 and illustrating amodification of the apparatus of FIG. 1 employing special lightposition-sensing photo-detectors 68 and 70 which are capable ofdetermining both the intensity and the angular position of scatterradiation. These are silicon barrier photo diodes ofthe type which arecommercially available for instance from United Detector Technology ofSanta Monica, Calif., the name of the product being designated as LightPosition Sensing Photo-Detectors. The photo detectors 68 and 70 aredriven by a direct current voltage source indicated at 72. The currentfrom source 72 enters the detectors 68 and 70 at the center detectorterminals 74 and 76. The current passes through either or both of thedetectors and is emitted at the end terminals 78 and 80, or at the endterminals 82 and 84, the ratio of the current through the different endterminals of each detector device is determined by the position of theradiation striking the device. For instance, if the radiation strikesthe device 70, and if the radiation position is closer to the terminal84 than it is to the terminal 80, there will be a greater current fromthe terminal 84 than there is from the terminal 80. The currents fromterminals 82 and 84 are returned to the source 72 through a loadresistor 86 to ground and from ground through a load resistor 88.Similarly, the currents from terminals 78 and 80 are returned through aload resistor 90 to ground and thus through load resistor 88 to thesource 72. The resistance values of load resistors 86 and 90 arepreferably equal and the relative values of the load currents throughthose resistors are measured in terms of voltage drops in a differentialamplifier 92 to provide a scatter angle signal fed to a detection andrecording circuit 46A.

Similarly, the intensity of the scatter illumination is detected interms of the voltage drop across the common load resistor 88 by anamplifier 94, the output of which is also supplied to the circuit 46A.Thus, the arrangement of FIG. 5 does not simply record scatter radiationin specified ranges of scatter angles, but rather it records theintensity of the scatter radiation and indicates the angle at which thatradiation occurs. This provides a particularly valuable analysis toolwhere the significant range of scatter angle is not previously known.

In accordance with the present invention it has been determined that anew method may be followed, employing the apparatus described above, forrapidly analyzing samples of particles, and particularly biologicalsamples of cells such as blood cells. As mentioned above, differentconditions of biological cells may control the amount of dye whichindividual cells will pick up when the cell sample is exposed to a dye.For instance, when a mixture oflive cells and dead cells is exposed toTrypan blue dye, the dead cells take up the dye to a significant extent,while the live cells do not.

In a specific example of the practice of this method, the method may beemployed to determine the feasibility of an organ transplant from oneindividual to another by checking for compatibility of blood cells. Forsuch a compatibility test, lymphocytes may be extracted from the bloodof a donor to be matched with the sera of potential recipients. Toeffect this match the lymphocytes are diluted in 0.85 percent sodiumchloride solution or an appropriate nutrient medium to a concentrationof5 million cells per milliliter and added to the diluted serum of thepotential donor. The mixture is then incubated at a temperature in therange from to Centrigrade for approximately minutes. The incubation isthen stopped by placing the samples on ice. Immediately beforeexamination a freshly prepared Trypan blue aqueous solution containing0.24 percent Trypan blue and 0.85 percent sodium chloride is added tothe sample in a volume equal to percent of the sample volume so as toincrease the sample volume by about 50 percent. The final suspensionshould have a cell concentration in the range of one million cells, orless, per milliliter of solution to provide reasonable assurance thatthe cells will pass through the apparatus one at a time..The sample isthencaused to pass through the apparatus as described above inconnection with FIG. 1, and the optical absorption of the cells isdetected concurrently with the optical scatter over a range from aboutone degree to about thirty degrees away from the axis of the beam. Thestained cells, which provide a large absorption signal, are indicated asdead, and the unstained cells, which provide a high scatter signal, anda low absorption signal are indicated as live. A high count of deadcells in this compatibility analysis indicates a lack of compatibilitybetween donor and recipient for the purpose of transplants.

For this analysis, a helium neon laser emitting illumination at 6300angstroms may be employed. Other dyes may be alternatively used for thislive-dead cell analysis method. For instance, nigrosine dyes are usefulfor this purpose.

FIG. 6 illustrates an essential step in the preferred method of theproduction of the optical chamber member 10 of the apparatus of thepresent invention. In practicing this method, a piece of glass tubinghaving inside and outside diameters of the desired size for the finishedoptical measurement portion of the tubular optical chamber is cut to alength which is somewhat greater than twice the desired length of thefinished optical chamber. The tubing must be substantially free of anycracks, scratches or blemishes. The glass tubing is then heateduniformly around the central portion ofthe axial length thereof to thesoftening temperature of the glass. This may be simply accomplished byrotating the tubing while the central portion of the-tubing is held in agas flame.

Next, the central bore of the tubing is placed under pressure. Thiscauses enlargement, or blowing out, of the central bore of the tubing atthe heated central portion thereof, as shown at 96 in FIG. 6.

When the proper degree of enlargement has been achieved, the pressure isreleased and the tubing is allowed to cool and harden. It is then cutapart through the centerline as indicated at 98. The two resultantseparate pieces 10A and 108 can each be used as optical chambers in anapparatus such as previously described in connection with FIG. 1 afterthe next step, As the final step, in order to provide a liquid-tightseal at the ends of each chamber, both ends of each of the glass parts10A and 10B are preferably ground flat and mutually parallel toeliminate the slight inaccuracies of the glass cutting procedure.

There are a number of useful variations in the process described justabove. For instance, the central bore of the tubing may be placed underpressure while the tubing is being heated so that the enlargement of thetubing occurs at the earliest possible moment when the heating hasprogressed sufficiently to soften the glass. Also, it has been found tobe quite practical and desirable to perform the heating and pressurizingsteps at spaced axial positions along an extended length of glasstubing. All of the tube cutting operations are then done after theenlargements have been formed, cutting at each enlargement and midwaybetween adjacent enlargements to thereby obtain a yield of two opticalchambers for each tubing enlargement.

It has been found that the above method of production ofthe opticalchambers is very simple and inexpensive and also very satisfactory. Ithas been determined that the changes in the diameter in the resultantenlarged funnel-shaped end 24 of the central bore 26 is substantially anexponential function. That is, the change in the diameter of the centralbore is substantially an exponential function of the displacement alongthe axis of the tube at which the diameter is measured. This provides agraded change in the bore diameter which is very valuable and useful inpromoting smooth and non-turbulent liquid flow in the critical borenarrowing region where the sheath liquid is reducing the diameter of theparticle carrying liquid stream.

While this invention has been shown and described in connection withparticular preferred embodiments, various alterations and modificationswill occur to those skilled in the art. Accordingly, the followingclaims are intended to define the valid scope of this invention over theprior art, and to cover all changes and modifications falling within thetrue spirit and valid scope of this invention.

We claim:

1. An apparatus for rapid optical measurement of the characteristics ofsmall particles such as blood cells while the particles are suspended ina liquid,

comprising a source of light,

a cylindrical tube member defining an optical chamber,

said tube member being comprised of a material which transmits lightfrom said source,

means for directing said light into one side of said tube member in abeam substantially converging at the center of said optical chamber whenviewed in a direction perpendicular to the axis of said tube memberdefining said chamber,

means for moving the particle suspending liquid through said tube memberin a thin stream to cause the particles therein to pass in sequencethrough said light beam one by one,

at least one photoresponsive pick up element for detecting lightscattered by said particles positioned on the side of said tube memberopposite to said light source and displaced away from the direct path ofsaid light beam through said tube member in a direction parallel to theaxis of said tube member,

said light source comprising a source of monochromatic light,

a second photoresponsive pick-up element in addition to said scatteredlight detecting photoresponsive pick-up element,

a spectral filtering element positioned to filter the light received bysaid second photoresponsive pickup element,

said spectral filtering element being selected to exclude light at thewave length of said light source and to pass light to the associatedphotoresponsive pick-up element at a wave length for which the particlesunder investigation emit fluorescent radiation in the presence ofasignificant particle characteristic to be detected,

and optical means substantially surrounding the circumference of saidcylindrical tube member optical chamber for gathering fluorescentradiation directed radially outwardly from the particles and fordirecting the fluorescent radiation through said spectral filteringelement to said second photoresponsive pick-up element.

2. Apparatus as claimed in claim 1 wherein there is provided a thirdphotoresponsive pick-up element positioned outside of said chamber and asecond spectral filtering element positioned to filter the lightreceived by said third photoresponsive pick-up element,

said second spectral filtering element being selected to exclude lightat the wave length of said light source and at the wave length passed bysaid firstmentioned spectral filtering element and operable to passlight to the associated photoresponsive pick-up element at a wave lengthfor which the particles under investigation emit fluorescent radiationin the presence of a second significant particle characteristic to bedetected.

3. Apparatus as claimed in claim 2 wherein said optical means forgathering and directing fluorescent radiation includes a dichroic mirroroperable to reflect fluorescent radiation to one of said spectralfiltering elements and the photoresponsive pick-up element associatedtherewith,

and operable to transmit fluorescent radiation to the other one of saidspectral filtering elements and the photoresponsive pick-up elementassociated therewith.

4. An apparatus for rapid optical measurement of the characteristics ofsmall particles such as blood cells while the particles are suspended ina liquid,

comprising a source of substantially monochromatic light,

a cylindrical tube member defining an optical chamber,

said tube member being comprised of a material which transmits lightfrom said source,

means for directing said light into one side of said tube member in abeam substantially converging at the center of said optical chamber whenviewed in a direction perpendicular to the axis of said tube memberdefining said chamber,

means for moving the particle suspending liquid through said tube memberin a thin stream to cause the particles therein to pass in sequencethrough said light beam one by one,

at least one photoresponsive pick-up element positioned outside of saidtube member and displaced away from the direct path of said light beamthrough said tube member,

said pick-up element being operable to respond to fluorescent radiationemitted by said particles in the presence of said light beam,

a spectral filtering element positioned to filter the light received bysaid pick-up element,

said spectral filtering element being selected to exclude light at thewave length of said light source and to pass light to said pick-upelement at a wave length for which the particles under investigationemit fluorescent radiation in the presence of a sig nificant particlecharacteristic to be detected,

and optical means substantially surrounding the circumference of saidoptical chamber and operable to gather fluorescent radiation directedradially outwardly through the walls of said optical chamber from theparticles and for directing the fluorescent radiation through saidspectral filtering element to said photoresponsive pick-up element.

5. Apparatus as claimed in claim 4 wherein there is provided a secondphotoresponsive pick-up element positioned in alignment with the lightbeam on the side of said housing opposite to said light source toreceive the unscattered illumination from the light beam.

6. Apparatus as claimed in claim 4 wherein a second photoresponsivepick-up element is positioned outside of said tube member to receivefluorescent radiation from the particles gathered by said optical meansfor gathering fluorescent radiation, a second spectral filtering elementpositioned to filter the light received by said second pick-up element,

said second spectral filtering element being selected to exclude lightat the wave length of said light source and to pass light to said secondpick-up element at a wave length for which the particles underinvestigation emit fluorescent radiation in the presence of asignificant particle characteristic to be detected which is differentfrom the characteristic detected by fluorescent radiation received bysaid previously mentioned pick-up element.

7. Apparatus as claimed in claim 6 wherein a dichroic mirror is providedand positioned between said optical means for gathering fluorescentradiation from the particles and said spectral filtering elements,

said dichroic mirror being operable to reflect fluorescent radiation toone of said spectral filtering elements and the photoresponsive pick-upelement associated therewith,

and said dichroic mirror being operable to transmit fluorescentradiation to the other one of said spectral filtering elements and thephotoresponsive pick-up element associated therewith.

8. Apparatus as claimed in claim 4 wherein said means for moving theparticle suspending liquid through said tube member in a thin streamincludes means for providing a sheath of liquid flowing in an annularconfiguration through said tube member and surrounding said thin streamto thereby confine the thin stream of particle suspending liquid to adimension smaller than the interior diameter of said tube member.

9. Apparatus as claimed in claim 4 wherein said light source is a laser.

10. Apparatus as claimed in claim 9 wherein said laser is an argon ionlaser.

11. An apparatus for rapid optical measurement of the characteristics ofsmall particles such as blood cells while the particles-are suspended ina liquid comprising a cylindrical tubular housing defining an opticalchamber,

a source of substantially monochromatic light,

said optical chamber being comprised of a material which transmits lightfrom said source,

means for moving the particle suspending liquid through said opticalchamber in a thin narrow stream to thereby convey the particles insequence through the light beam one by one,

means for directing light from said light source into one side of saidoptical chamber to intersect with the thin stream carrying the particlestherein,

said means for directing said light being operable in cooperation withsaid side of said optical chamber to converge said light beam into asubstantially elliptical shape in a plane transverse to the light beamand at the point of intersection with the position of the thin stream ofparticles,

the major axis of the elliptical shape of said beam being substantiallyperpendicular to the direction of the stream of particles and thedimension of said beam at said major axis being substantially greaterthan the transverse dimension of said particle stream,

at least one photoresponsive pick-up element positioned outside of saidoptical chamber,

said pick-up element being operable to respond to fluorescent radiationemitted by said particles in the presence of said light beam,

a spectral filtering element positioned to filter the light received bysaid pick-up element,

said spectral filtering element being selected to exclude light at thewave length of said light source and to pass light to said pick-upelement at a wave length for which the particles under investigationemit fluorescent radiation in the presence of a significant particlecharacteristic to be detected,

and optical means substantially surrounding the circumference of saidoptical chamber and operable to gather fluorescent radiation directedradially outwardly through the walls of said optical chamber from theparticles and for directing the fluorescent radiation through saidspectral filtering element to said photoresponsive pick-up element.

12. Apparatus as claimed in claim 11 wherein said means for directinglight from said light source comprises a substantially cylindrical lensdevice.

13. A method for rapidly detecting biological cells having differentproperties including the steps of exposing a solution containing thecells to be analyzed to a dye which is taken up differently by cellshaving different characteristics,

moving the particle suspending liquid solution in a thin stream toconvey the particles one by one through a cylindrical tubular opticalchamber while directing light from a substantially monochromatic lightsource into one side of the chamber to inter- I sect with the thinstream of particles in a narrow beam,

the narrow beam converging at the stream of particles,

gathering fluorescence radiation signals emitted by the stained cellswhen excited by the light beam and radiated radially outwardly from thecircumference of the housing, 7

detecting said radiation signals by means of at least onephotoresponsive pick-up element positioned outside of the housing,

and filtering the fluorescence radiation signals directed to the pick-upelement to exclude light at the wave length of said light source and topass light to the pick-up element at a wave length for which theparticles under investigation emit fluorescent radiation in the presenceof a significant particle characteristic to be detected.

222233? 1 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PatentNo. 8,744 mzLJ-fi Inventor(s) MITCHELL FRIEDMAN, mnm A I v v T T INGEIIt is certified'that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

COlumnl, lines 4 and 5, "U.S. Pat. No. 3,687,553 issued 8/29/72 shouldread -U.S. Pat. No. 3,705,771 issued l2/l2/72; lastline, "sidderent"should read different--.

Column 2, line 17, "is" should read -has-;

line 43, "of" should read -on--.

Column 3, line 37, "an" should read --andline6'4, "particle" should read--particles--.

Column 4, line 39, "signlas" should read --signals--;

line 45, iverges" should read -diverges--. Column 5, lines 5 and 6,"'abosrption" should read -absorption--;

line 11, "a" should read --the--. Column 6, line 5, "diamter" shouldread --diameter-; line 13, "strea" should re'ad-stream--'.

Column 7, line 5, "refelcted" should read -reflected-;

line 26, "compounds" should read comp0nents--.

Column 8, lines 38 and '39, "preferably" should read --preferable lines51 and 52, "surface 3o" should read --source 30-- lines 55 and 56,"oabservation" should read observation-; 7 line59, "radii-ion shouldread -radiation--.

' Signed and sealed this 11th day of June 19714..

(SEAL) Attest:

EDWARD M.FLETCHER,JR. T I c. MARSHALL mum Attesting Officer vCommissioner of Patents

1. An apparatus for rapid optical measurement of the characteristics ofsmall particles such as blood cells while the particles are suspended ina liquid, comprising a source of light, a cylindrical tube memberdefining an optical chamber, said tube member being comprised of amaterial which transmits light from said source, means for directingsaid light into one side of said tube member in a beam substantiallyconverging at the center of said optical chamber when viewed in adirection perpendicular to the axis of said tube member defining saidchamber, means for moving the particle suspending liquid through saidtube member in a thin stream to cause the particles therein to pass insequence through said light beam one by one, at least onephotoresponsive pick-up element for detecting light scattered by saidparticles positioned on the side of said tube member opposite to saidlight source and displaced away from the direct path of said light beamthrough said tube member in a direction parallel to the axis of saidtube member, said light source comprising a source of mono-chromaticlight, a second photoresponsive pick-up element in addition to saidscattered light detecting photoresponsive pick-up element, a spectralfiltering element positioned to filter the light received by said secondphotoresponsive pick-up element, said spectral filtering element beingselected to exclude light at the wave length of said light source and topass light to the associated photoresponsive pick-up element at a wavelength for which the particles under investigation emit fluorescentradiation in the presence of a significant particle characteristic to bedetected, and optical means substantially surrounding the circumferenceof said cylindrical tube member optical chamber for gatheringfluorescent radiation directed radially outwardly from the particles andfor directing the fluorescent radiation through said spectral filteringelement to said second photoresponsive pick-up element.
 2. Apparatus asclaimed in claim 1 wherein there is provided a third photoresponsivepick-up element positioned outside of said chamber and a second spectralfiltering element positioned to filter the light received by said thirdphotoresponsive pick-up element, said second spectral filtering elementbeing selected to exclude light at the wave length of said light sourceand at the wave length passed by said first-mentioned spectral filteringelement and operable to pass light to the associated photoresponsivepick-up element at a wave length for which the particles underinvestigation emit fluorescent radiation in the presence of a secondsignificant particle characteristic to be detected.
 3. Apparatus asclaimed in claim 2 wherein said optical means for gathering anddirecting fluorescent radiation includes a dichroic mirror operable toreflect fluorescent radiation to one of said spectral filtering elementsand the photoresponsive pick-up element associated therewith, andoperable to transmit fluorescent radiation to the other one of saidspectral filtering elements and the photoresponsive picK-up elementassociated therewith.
 4. An apparatus for rapid optical measurement ofthe characteristics of small particles such as blood cells while theparticles are suspended in a liquid, comprising a source ofsubstantially monochromatic light, a cylindrical tube member defining anoptical chamber, said tube member being comprised of a material whichtransmits light from said source, means for directing said light intoone side of said tube member in a beam substantially converging at thecenter of said optical chamber when viewed in a direction perpendicularto the axis of said tube member defining said chamber, means for movingthe particle suspending liquid through said tube member in a thin streamto cause the particles therein to pass in sequence through said lightbeam one by one, at least one photoresponsive pick-up element positionedoutside of said tube member and displaced away from the direct path ofsaid light beam through said tube member, said pick-up element beingoperable to respond to fluorescent radiation emitted by said particlesin the presence of said light beam, a spectral filtering elementpositioned to filter the light received by said pick-up element, saidspectral filtering element being selected to exclude light at the wavelength of said light source and to pass light to said pick-up element ata wave length for which the particles under investigation emitfluorescent radiation in the presence of a significant particlecharacteristic to be detected, and optical means substantiallysurrounding the circumference of said optical chamber and operable togather fluorescent radiation directed radially outwardly through thewalls of said optical chamber from the particles and for directing thefluorescent radiation through said spectral filtering element to saidphotoresponsive pick-up element.
 5. Apparatus as claimed in claim 4wherein there is provided a second photoresponsive pick-up elementpositioned in alignment with the light beam on the side of said housingopposite to said light source to receive the unscattered illuminationfrom the light beam.
 6. Apparatus as claimed in claim 4 wherein a secondphotoresponsive pick-up element is positioned outside of said tubemember to receive fluorescent radiation from the particles gathered bysaid optical means for gathering fluorescent radiation, a secondspectral filtering element positioned to filter the light received bysaid second pick-up element, said second spectral filtering elementbeing selected to exclude light at the wave length of said light sourceand to pass light to said second pick-up element at a wave length forwhich the particles under investigation emit fluorescent radiation inthe presence of a significant particle characteristic to be detectedwhich is different from the characteristic detected by fluorescentradiation received by said previously mentioned pick-up element. 7.Apparatus as claimed in claim 6 wherein a dichroic mirror is providedand positioned between said optical means for gathering fluorescentradiation from the particles and said spectral filtering elements, saiddichroic mirror being operable to reflect fluorescent radiation to oneof said spectral filtering elements and the photoresponsive pick-upelement associated therewith, and said dichroic mirror being operable totransmit fluorescent radiation to the other one of said spectralfiltering elements and the photoresponsive pick-up element associatedtherewith.
 8. Apparatus as claimed in claim 4 wherein said means formoving the particle suspending liquid through said tube member in a thinstream includes means for providing a sheath of liquid flowing in anannular configuration through said tube member and surrounding said thinstream to thereby confine the thin stream of particle suspending liquidto a dimension smaller than the interior diameter of said tube member.9. Apparatus as claImed in claim 4 wherein said light source is a laser.10. Apparatus as claimed in claim 9 wherein said laser is an argon ionlaser.
 11. An apparatus for rapid optical measurement of thecharacteristics of small particles such as blood cells while theparticles are suspended in a liquid comprising a cylindrical tubularhousing defining an optical chamber, a source of substantiallymonochromatic light, said optical chamber being comprised of a materialwhich transmits light from said source, means for moving the particlesuspending liquid through said optical chamber in a thin narrow streamto thereby convey the particles in sequence through the light beam oneby one, means for directing light from said light source into one sideof said optical chamber to intersect with the thin stream carrying theparticles therein, said means for directing said light being operable incooperation with said side of said optical chamber to converge saidlight beam into a substantially elliptical shape in a plane transverseto the light beam and at the point of intersection with the position ofthe thin stream of particles, the major axis of the elliptical shape ofsaid beam being substantially perpendicular to the direction of thestream of particles and the dimension of said beam at said major axisbeing substantially greater than the transverse dimension of saidparticle stream, at least one photoresponsive pick-up element positionedoutside of said optical chamber, said pick-up element being operable torespond to fluorescent radiation emitted by said particles in thepresence of said light beam, a spectral filtering element positioned tofilter the light received by said pick-up element, said spectralfiltering element being selected to exclude light at the wave length ofsaid light source and to pass light to said pick-up element at a wavelength for which the particles under investigation emit fluorescentradiation in the presence of a significant particle characteristic to bedetected, and optical means substantially surrounding the circumferenceof said optical chamber and operable to gather fluorescent radiationdirected radially outwardly through the walls of said optical chamberfrom the particles and for directing the fluorescent radiation throughsaid spectral filtering element to said photoresponsive pick-up element.12. Apparatus as claimed in claim 11 wherein said means for directinglight from said light source comprises a substantially cylindrical lensdevice.
 13. A method for rapidly detecting biological cells havingdifferent properties including the steps of exposing a solutioncontaining the cells to be analyzed to a dye which is taken updifferently by cells having different characteristics, moving theparticle suspending liquid solution in a thin stream to convey theparticles one by one through a cylindrical tubular optical chamber whiledirecting light from a substantially monochromatic light source into oneside of the chamber to intersect with the thin stream of particles in anarrow beam, the narrow beam converging at the stream of particles,gathering fluorescence radiation signals emitted by the stained cellswhen excited by the light beam and radiated radially outwardly from thecircumference of the housing, detecting said radiation signals by meansof at least one photoresponsive pick-up element positioned outside ofthe housing, and filtering the fluorescence radiation signals directedto the pick-up element to exclude light at the wave length of said lightsource and to pass light to the pick-up element at a wave length forwhich the particles under investigation emit fluorescent radiation inthe presence of a significant particle characteristic to be detected.